An essential step to understand the function of a protein is to identify other proteins with which it interacts. One of the most effective methods for identifying and measuring biologically important protein-protein interactions is the yeast two-hybrid system. The goal of this project is to implement a high-throughput yeast two-hybrid approach to identify the binary interactions among most of the proteins encoded by the Drosophila genome. The resulting protein interaction map will be a valuable resource for generating and testing hypotheses about protein and pathway function. Because many molecular pathways are evolutionarily conserved, the Drosophila interaction map will provide a foundation for understanding homologous protein networks in humans and other organisms. To construct the map, the protein-coding regions of most Drosophila genes will be amplified using gene-specific primer pairs. The resulting set of PCR products will provide a starting point for efficient transfer of the open reading frames (ORFs) into a number of useful vectors. and thus will be a valuable resource for a variety of projects. For this project, the Drosophila ORFs will be transferred into two yeast two-hybrid vectors, one for expressing DNA-binding domain (DBD) fusions and one for expressing activation domain (AD) fusions. The constructs will be introduced into specialized yeast strains to create two arrays of yeast. To test for interactions between members of the two arrays, reporter gene activity will be assessed after the arrays are mated using a high-throughput strategy developed in this laboratory. The protein interaction data will be placed into a publicly accessible database, designed specifically to enable extraction of useful biological information from yeast two-hybrid data. When combined with sequence information and data from other functional genomics approaches, the interaction data will provide a powerful resource to help researchers understand how groups of genes work together to mediate complex biological processes.